Two efficient recombination systems were combined to produce a versatile method for chromosomal engineering that obviates the need to prepare double-stranded DNA (dsDNA) recombination substrates. A synthetic "targeting oligonucleotide" is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annealase. This oligonucleotide contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a "payload plasmid" that contains a cognate recombination site and a selectable marker. The targeting oligonucleotide and payload plasmid are cotransformed into a RecT- and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate gene knockouts, promoter replacements, or C-terminal tags. This new system is called ORBIT (for "oligonucleotide-mediated recombineering followed by Bxb1 integrase targeting") and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions, or fusions in a bacterial chromosome. We demonstrate the utility of this "drag and drop" strategy by the construction of insertions or deletions in over 100 genes in Mycobacterium tuberculosis and M. smegmatis IMPORTANCE We sought to develop a system that could increase the usefulness of oligonucleotide-mediated recombineering of bacterial chromosomes by expanding the types of modifications generated by an oligonucleotide (i.e., insertions and deletions) and by making recombinant formation a selectable event. This paper describes such a system for use in M. smegmatis and M. tuberculosis By incorporating a single-stranded DNA (ssDNA) version of the phage Bxb1 attP site into the oligonucleotide and coelectroporating it with a nonreplicative plasmid that carries an attB site and a drug selection marker, we show both formation of a chromosomal attP site and integration of the plasmid in a single transformation. No target-specific dsDNA substrates are required. This system will allow investigators studying mycobacterial diseases, including tuberculosis, to easily generate multiple mutants for analysis of virulence factors, identification of new drug targets, and development of new vaccines.

Current methods for genome engineering in mycobacteria rely on relatively inefficient recombination systems that require the laborious construction of a long double-stranded DNA substrate for each desired modification. We combined two efficient recombination systems to produce a versatile method for high-throughput chromosomal engineering that obviates the need for the preparation of double-stranded DNA recombination substrates. A synthetic targeting oligonucleotide is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annelase. This oligo contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a payload plasmid that contains a cognate recombination site and selectable marker. The targeting oligo and payload plasmid are co-transformed into a RecT- and Int- expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate knockouts and promoter replacements, or to fuse the C-terminal-encoding regions of target genes with tags of various functionalities. This new system is called ORBIT (Oligo-mediated Recombineering followed by Bxb1 Integrase Targeting) and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions or fusions in a target bacterium. We demonstrate the utility of ORBIT by the construction of insertions or deletions in over 100 genes in M. tuberculosis and M. smegmatis. The report describes the first genetic engineering technique for making selectable chromosomal fusions and deletions that does not require the construction of target- or modification-specific double-stranded DNA recombination substrates.

Nitric oxide contributes to protection from tuberculosis. It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis growth, which prevents subsequent pathological inflammation. In contrast, we report that nitric oxide primarily protects mice by repressing an interleukin-1- and 12/15-lipoxygenase-dependent neutrophil recruitment cascade that promotes bacterial replication. Using M. tuberculosis mutants as indicators of the pathogen's environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports M. tuberculosis growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes tuberculosis in patients. The human 12/15-lipoxygenase orthologue, ALOX12, is expressed in cavitary tuberculosis lesions; the abundance of its products correlates with the number of airway neutrophils and bacterial burden and a genetic polymorphism that increases ALOX12 expression is associated with tuberculosis risk. These data suggest that M. tuberculosis exploits neutrophilic inflammation to preferentially replicate at sites of tissue damage that promote contagion.

Tn-Seq is an experimental method for probing the functions of genes through construction of complex random transposon insertion libraries and quantification of each mutant's abundance using next-generation sequencing. An important emerging application of Tn-Seq is for identifying genetic interactions, which involves comparing Tn mutant libraries generated in different genetic backgrounds (e.g. wild-type strain versus knockout strain). Several analytical methods have been proposed for analyzing Tn-Seq data to identify genetic interactions, including estimating relative fitness ratios and fitting a generalized linear model. However, these have limitations which necessitate an improved approach. We present a hierarchical Bayesian method for identifying genetic interactions through quantifying the statistical significance of changes in enrichment. The analysis involves a four-way comparison of insertion counts across datasets to identify transposon mutants that differentially affect bacterial fitness depending on genetic background. Our approach was applied to Tn-Seq libraries made in isogenic strains of Mycobacterium tuberculosis lacking three different genes of unknown function previously shown to be necessary for optimal fitness during infection. By analyzing the libraries subjected to selection in mice, we were able to distinguish several distinct classes of genetic interactions for each target gene that shed light on their functions and roles during infection.

Little is known about iron efflux transporters within bacterial systems. Recently, the participation of Bacillus subtilis PfeT, a P1B4-ATPase, in cytoplasmic Fe(2+) efflux has been proposed. We report here the distinct roles of mycobacterial P1B4-ATPases in the homeostasis of Co(2+) and Fe(2+) Mutation of Mycobacterium smegmatis ctpJ affects the homeostasis of both ions. Alternatively, an M. tuberculosis ctpJ mutant is more sensitive to Co(2+) than Fe(2+), whereas mutation of the homologous M. tuberculosis ctpD leads to Fe(2+) sensitivity but no alterations in Co(2+) homeostasis. In vitro, the three enzymes are activated by both Fe(2+) and Co(2+) and bind 1 eq of either ion at their transport site. However, equilibrium binding affinities and activity kinetics show that M. tuberculosis CtpD has higher affinity for Fe(2+) and twice the Fe(2+)-stimulated activity than the CtpJs. These parameters are paralleled by a lower activation and affinity for Co(2+) Analysis of Fe(2+) and Co(2+) binding to CtpD by x-ray absorption spectroscopy shows that both ions are five- to six-coordinate, constrained within oxygen/nitrogen environments with similar geometries. Mutagenesis studies suggest the involvement of invariant Ser, His, and Glu residues in metal coordination. Interestingly, replacement of the conserved Cys at the metal binding pocket leads to a large reduction in Fe(2+) but not Co(2+) binding affinity. We propose that CtpJ ATPases participate in the control of steady state Fe(2+) levels. CtpD, required for M. tuberculosis virulence, is a high affinity Fe(2+) transporter involved in the rapid response to iron dyshomeostasis generated upon redox stress.

The physiological functions of macrophage, which plays a central role in the pathogenesis of tuberculosis, depend on its redox state. System xc-, a cystine-glutamate transporter, which consists of xCT and CD98, influences many ROS-dependent pathways by regulating the production of the antioxidant glutathione. xCT's ability to alter this critical host redox balance by increasing the glutathione synthesis aspect of phagocyte physiology suggested that it might influence tuberculosis pathogenesis. In this study, we found that the xCT expression was increased in peripheral blood monocyte of active tuberculosis. xCT expression in macrophage was induced by Mycobacterium tuberculosis (Mtb) through TLR2/Akt- and p38-dependent signaling pathway. Importantly, xCT deficiency conferred protection against tuberculosis, as xCT knock out mice displayed increased Mtb load and reduced pulmonary pathology in lung compared to wild type mice. xCT disruption enhanced the mycobateriacidal activity of macrophage through increasing the mycothiol oxidation. Importantly, chemical inhibition of xCT with sulfasalazine, a specific xCT inhibitor that is already approved by the FDA for treatment of inflammatory bowel disease, produces similar protective effects in vivo and in vitro, indicating xCT might be a novel and useful target for host-directed TB treatment strategy.